The present invention relates to a system for the production of proteins in insect larvae. In particular, the invention encompasses the production of proteins of interest in Drosophila and Medfly larvae.
Protein production systems, in which polypeptides or proteins of interest are produced in recombinant organisms or cells, are the backbone of commercial biotechnology. The earliest systems, based on bacterial expression in hosts such as E. coli, have been joined by systems based on eukaryotic hosts, in particular mammalian cells in culture, insect cells both in culture and in the form of whole insects, and transgenic mammals such as sheep and goats.
Prokaryotic cell culture systems are easy to maintain and cheap to operate. However, prokaryotic cells are not capable of post-translational modification of eukaryotic proteins. Moreover, many proteins are incorrectly folded, requiring specific procedures to refold them, which adds to the cost of production.
Eukaryotic cell culture systems have been described for a number of applications. For example, mammalian cells are capable of post-translational modification, and generally produce proteins which are correctly folded and soluble. The chief disadvantages of mammalian cell systems include the requirement for specialised and expensive culture facilities, and the risk of infection, which can lead to loss of the whole culture.
Plant production systems may be used for protein expression, and may achieve high-yield production. However, transgenic plants crops are difficult to contain, raising the risk of contamination of the environment with genetically manipulated material.
Insect cells are also used for polypeptide expression. The most widespread expression system used in insect cells is based on baculovirus vectors. A baculovirus expression vector is constructed by replacing the polyhedrin gene of baculovirus, which encodes a major structural protein of the baculovirus, with a heterologous gene, under the control of the strong native polyhedrin promoter. Cultured insect host cells are infected with the recombinant virus, and the protein produced thereby can be recovered from the cells themselves or from the culture medium if suitable secretion signals are employed. These systems also, however, suffer from problems associated with infection of the culture and the requirement for specialised culture facilities.
Organism based expression systems avoid many of the infection disadvantages and are easier to grow than cell cultures. For instance, the use of virus vectors such as baculovirus allows infection of entire insects, which have fewer requirements for special growth conditions. Large insects, such as silk moths, provide a high yield of heterologous protein. The protein can be extracted from the insects according to conventional extraction techniques.
Also known are techniques based on expression of proteins of interest in mammals such as goats and sheep, under the control of milk protein expression control sequences such that they are expressed in milk: Such techniques have great potential advantages, but are expensive due to the requirement for isolation of endogenous mammalian viruses, prions and proteins from the final product. Moreover, the cost of generating and keeping large transgenic animals is high.
The use of insect larvae, those of Trichoplusa ni, have been proposed for use in protein production systems (Pham et al., 1999 Biotech Bioeng 62:175–182). However, such systems have only been suggested in combination with viral vector technology based around baculoviruses.
Where proteins are intended for dietary or pharmaceutical use, the use of bacterial systems and/or viral vectors is undesirable. There is therefore a requirement in the art for a protein production system which is both robust and scaleable, as whole organism based systems are, and free from virus-based vectors, as well as inexpensive to operate in a contained environment.